Filamentous Fungi and Mycotoxins in Cheese:

A Review
Nolwenn Hymery, Valérie Vasseur, Monika Coton, Jérôme Mounier, Jean-Luc Jany, Georges Barbier, and Emmanuel Coton

Abstract: Important fungi growing on cheese include Penicillium, Aspergillus, Cladosporium, Geotrichum, Mucor, and
Trichoderma. For some cheeses, such as Camembert, Roquefort, molds are intentionally added. However, some contami-
nating or technological fungal species have the potential to produce undesirable metabolites such as mycotoxins. The most
hazardous mycotoxins found in cheese, ochratoxin A and aflatoxin M1, are produced by unwanted fungal species either via
direct cheese contamination or indirect milk contamination (animal feed contamination), respectively. To date, no human
food poisoning cases have been associated with contaminated cheese consumption. However, although some studies
state that cheese is an unfavorable matrix for mycotoxin production; these metabolites are actually detected in cheeses at
various concentrations. In this context, questions can be raised concerning mycotoxin production in cheese, the biotic
and abiotic factors influencing their production, mycotoxin relative toxicity as well as the methods used for detection and
quantification. This review emphasizes future challenges that need to be addressed by the scientific community, fungal
culture manufacturers, and artisanal and industrial cheese producers.
Keywords: Mold, filamentous fungi, mycotoxins, cheeses, diversity, mycotoxin toxicity, mycotoxin biosynthesis, myco-
toxin ecological role, mycotoxin control

Introduction
Many food spoilage fungi belonging to the genera Aspergillus,
Fusarium, and Penicillium produce mycotoxins that are toxic for
vertebrate and other animal groups when introduced via a natural
route (food ingestion). Naturally present in ambient air, soil, and
crops (Yiannikouris and Jouany 2002), mycotoxigenic fungi are
considered to be among the most significant contaminants in foods
in terms of impact on public health, food safety, and the national
economy of several countries (Steyn 1995; Pitt 2000).
The term mycotoxin, derived from the Greek words “mykes”
meaning fungi or slime molds and “toxicum” referring to toxin,
was utilized for the first time in 1962 following a veterinary crisis
that resulted in the death of about 100000 young turkeys. These
birds had ingested contaminated peanut meal containing secondary
metabolites (aflatoxins [AFs]) produced by Aspergillus flavus (Blount
1961; Hundley 2001).
Mycotoxins are defined as secondary metabolites, hence
not directly essential for normal fungal growth, and are often
low-molecular-weight compounds. They are naturally occurring
molecules and are thought to confer a selective advantage to the
producer strain within complex ecosystems. Their role is to com-
municate and compete with other organisms or to inhibit competi-
MS 20131758 Submitted 11/26/2013, Accepted 12/2/2014. Authors are with
Laboratoire Universitaire de Biodiversité et d’Ecologie Microbienne, ESIAB, Technopôle
de Brest Iroise, Université de Brest, EA3882, 29280 Plouzané, France. Direct
inquiries to author Hymery (E-mail: nolwenn.hymery@univ-brest.fr).
This article was originally published on 23 June 2014. Subsequently Figure 2
was corrected to include a previously missing part. The corrected version was
published on 10 July 2014.

C 2014 Institute of Food Technologists®
tor growth and reproduction in the same trophic niche (Magan
and Aldred 2007; Fox and Howlett 2008). Early mycotoxin
production could also allow molds to rapidly colonize the envi-
ronment. These metabolites are commonly found in various food
and feed commodities. Indeed, molds are able to grow on a wide
range of substrates (including cereals, meats, nuts, cheeses, grapes,
coffee beans, apples, and derived products), at any stage of pro-
duction in the field, during postharvest storage, and under a wide
range of climatic conditions. Moreover, mycotoxins are known
to be resistant to industrial processing. It has been established that
mycotoxin production profiles may change under different fungal
growth conditions including substrate composition (Kokkonen
and others 2005a), ecophysiological factors including temperature,
water activity, pH or oxygen concentration, and biotic factors
including microbial interactions (Magan and Aldred 2007).
Analytical methods for identifying and quantifying mycotox-
ins in foods and feeds have been widely developed. Maximum
levels for AFs, deoxynivalenol, fumonisins, ochratoxin A (OTA),
patulin, T2 and HT-2 toxins, and zearalenone are regulated in
the EU (Commission Regulation [EC] Nr. 1881/2006) as well as
worldwide due to their well-characterized adverse health effects
after acute, short-term, and/or long-term exposure. In cheese,
aflatoxin M1 (AFM1) is the only mycotoxin for which maximum
levels (0.05 and 0.5 ppb in the milk used for cheese-making in the
EU and United States and China, respectively) have been set.
In cheese, the most hazardous mycotoxins are OTA and AFM1.
They are, respectively, produced by unwanted fungal species either
via direct cheese contamination (spoilage agents) or indirect con-
tamination of the milk used for cheese-making due to feed con-
tamination. Among fungal cheese spoilers, the most problematic

doi: 10.1111/1541-4337.12069 Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 437
Filamentous fungi and mycotoxins in cheese . . .
mycotoxigenic ones belong to Penicillium spp. as they are partic-
ularly well adapted for growth on and/or in the cheese matrix.
Filamentous fungi also play a central role during cheese-making.
Two well-known fungal ripening cultures, Penicillium camemberti
and Penicillium roqueforti, which play a significant role in ap-
pearance, texture, and flavor development of mold-ripened and
blue-veined cheeses, are also known for their ability to produce
several mycotoxins. For example, P. camemberti produces cyclopi-
azonic acid (CPA), while roquefortine C and mycophenolic acid
(MPA) are produced by P. roqueforti. Yet, these metabolites exhibit
lower toxicity than those reported above and are present at low
levels in cheese. Due to their long history of safe use in cheese in
the U.S.A. and EU (no cases of food poisoning associated with the
consumption of contaminated cheese were described in literature),
use of these fungal cultures is regarded as safe in their intended
conditions of use (cheese-making). In addition, these microorgan-
isms are granted a generally recognized as safe (GRAS) status by
the U.S Food and Drug Administration (FDA). However, in the
EU, while the European Food Safety Agency (EFSA) acknowl-
edged that there are no reports of adverse health effects for cheeses
produced with these microorganisms, the qualified presumption
of safety (QPS) status was not granted for these species. This was
mainly due to a lack of knowledge on mycotoxin production by
the strains intentionally used, the occurrence of these mycotoxins
in cheese and their toxicity (EFSA 2011). In this context, sci-
entific questions remain concerning the presence of mycotoxins
in cheese, their variability in terms of quality and quantity, rela-
tive toxicity levels, impact of biotic and abiotic factors on their
production as well as their control.
The aim of the review is to summarize the current knowledge
on filamentous fungi and mycotoxins in cheese. The paper deals
with fungal diversity, the main mycotoxins reported in cheeses,
mycotoxin toxicity, and their detection and quantification. The
biosynthetic pathways described to date in cheese-related species
as well as abiotic and biotic factors impacting mycotoxin produc-
tion in cheese are also presented. Finally, physical, chemical, and
biological methods potentially usable to prevent fungal growth
and indirectly eliminate or reduce mycotoxin levels in cheese are
discussed (from farm to fork).
Filamentous Fungi in Cheeses: The Good and the Bad
Few studies have extensively investigated fungal communities in
cheeses. Barrios and others (1998) provided a global view of mold
diversity in 52 commercial cheese susing a culture-dependent ap-
proach. Penicillium species were the most frequent (found in 63% of
cheeses), followed by Mucor spp. (27%), Geotrichum candidum (17%),
and 12 other fungal genera identified in 2 to 10% of the samples.
Such culture-dependent methods have also identified Penicillium
spp. as the prominent mold in sheep and goat cheeses from different
origins (Montagna and others 2004; Hayaloglu and Kirbag 2007;
Båth and others 2012). More recently, culture-independent meth-
ods have allowed to easily evaluate fungal (both undesirable and
technological) community dynamics in cheese (Jany and Barbier
2008). In this context, Flórez and Mayo (2006) used PCR-DGGE
to study the dynamics of Cabrales blue-veined cheese microbial
communities. In artisanal Cabrales cheese, they found a fungal
community composed of P. roqueforti, P. chrysogenum, or P. grise-
ofulvum that did not change during ripening. Concerning mold
surface-ripened cheeses, Arteau and Labrie (2010) recently studied
the fungal communities of Camembert and Brie cheeses using a
culture-independent T-RFLP method. In this study, Cladosporium
cladosporioides, G. candidum, M. racemosus, P. camemberti, P. caseicola,
P. chrysogenum, P. commune, and P. roqueforti filamentous fungal
438 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014
species were observed. Finally, use of both culture-dependent and
culture-independent methods allowed Panelli and others (2012)
to reveal the prominence of P. commune over other fungal species
such as Cladosporium spp. in Taleggio cheese, whereas in Fossa
cheese, the fungal community was dominated by Penicillium and
Aspergillus species (De Santi and others 2010).
Microorganisms play a central role during cheese-making, as
for other fermented products. When the primary microbiota in-
volved in acid production is mainly composed of starter lactic acid
bacteria (LAB), the secondary microbiota includes salt-tolerant
bacteria, yeasts, and filamentous fungi that mainly contribute to
organoleptic properties of cheeses. The role of filamentous fungi
is particularly important for both surface mold-ripened and core
mold-ripened cheeses. Indeed, functional features and degradation
of milk constituents, such as proteins and lipids, can lead to tex-
ture, flavor, and nutritional cheese quality improvement (Fox and
McSweeney 2004). Among the species encountered in cheeses,
P. camemberti, P. roqueforti, Mucor fuscus, M. lanceolatus, G. candidum,
Fusarium domesticum, Sporendonema casei, Scopulariopsis flava, and S.
fusca are the most common filamentous fungi used as ripening
cultures (Hermet and others 2012; Ropars and others 2012).
Filamentous fungi encountered in cheeses may originate from
raw materials such as milk or may be introduced during cheese-
making either from the environment or are deliberately inoculated
using commercial ripening cultures. Delavenne and others (2011)
showed that cow milk samples contained high fungal diversity with
up to 15 species in a single sample, whereas a maximum of 4 or 6
different species were recovered in goat and sheep milks, respec-
tively. These authors identified fungi belonging to the following
genera: Aspergillus, Chrysosporium, Cladosporium, Fusarium, Penicil-
lium, and Torrubiella. Vacheyrou and others (2011) concluded that
most fungal species found in cow milk originated from a transfer
from the stable to the milking parl and then to milk. Fungi oc-
curring in raw milk or in the cheese-making environment are not
very likely to persist and/or grow in cheese due to the biochem-
ical composition of cheese and the hurdle effect exerted by the
cheese microbiota. Industrial processes may also select certain fun-
gal strains/species resulting in a fungal community shift. Moreover,
raw milk heat treatment is often performed prior to manufactur-
ing and eliminates most fungi in cheeses where the presence of
molds is undesirable. Filamentous fungi contamination can also be
detrimental to cheese quality, causing appearance defects, off-flav,
and potentially toxic secondary metabolite production.
P. commune, P. palitans, P. nalgiovense, and P. verrucosum species are
the most frequent contaminants as suggested by Lund and others
(1995) in a study based on spoilage microbiota of hard, semi-
hard, and soft cheeses. Reports of P. verrucosum occurrence should,
however, be treated with care since a high number of P. verrucosum
isolates were reclassified as P. nordicum a species closely related to
P. verrucosum that is much more adapted to salt and protein-rich
substrates than P. nordicum (Larsen and others 2001). Fungal species
cannot always be classified as either technological or contaminant
species, since the same species can be used as ripening culture
in some cases and be undesirable in other contexts (for example,
P. roqueforti is the major ripening culture in blue-veined cheeses,
whereas it is a contaminant in other cheeses). In some cases, the
positive (technological species) or negative (spoilage organism or
putative mycotoxin producer) role is not always clear and has to be
investigated such as in the case of P. commune in Taleggio cheeses
(Panelli and others 2012).
In the near future, new high-throughput methodologies (such
as pyrosequencing) will certainly provide better insight into
cheese fungal communities. Among various filamentous fungi

C 2014 Institute of Food Technologists®
Filamentous fungi and mycotoxins in cheese . . .

Table 1–Examples of mycotoxins found in cheeses including blue mold cheese. ND: Not determined.

Secondary
Cheeses Mold species metabolites Technical analysis Range References Comments
Blue P. roqueforti Roquefortine, PR Thin-layer 3.4 μg/g, ND, ND Scott and others
toxin, chromatography (1977); Lund and
Isofumigaclavine A others (1995)
Blue P. roqueforti 100% penicillic acid, Thin-layer ND, ND van Egmond (1983)
37% PR toxin, chromatography
patulin
Blue-molded P. roqueforti Mycophenolic acid Thin-layer 90% 1 to 5 mg/kg Lafont and others
chromatography (1979)
Blue-molded P. roqueforti Patulin, penicillic acid, Thin-layer ND, ND, ND, 2.1 to 3.8 Erdogan and Sert
tulum cheese PR toxin, chromatography mg/kg (2004)
roquefortine
Blue veined P. roqueforti Penicillic acid Thin-layer ND Moubasher and others Production of
chromatography (1978) penicillic acid is
to be due to
P. carneum
(Boysen and
others 1996)
Blue OTA LC-ESI-MS/MS 0.2 to 0.3 μg/kg Dall’Asta and others
(2008)
Manchego P. roqueforti Roquefortine, Thin-layer ࣙ30 μg/kg López-Diaz and others
mycophenolic acid chromatography ࣙ 20 μg/kg (1996)
Cheddar P. roqueforti Penicillic acid, patulin Thin-layer ND, ND Olivigni and Production of
chromatography Bullerman (1977) penicillic acid is
to be due to
P. carneum
(Boysen and
others 1996)
Patulin was the
sole metabolite
produced by
P. paneum and
P. carneum but
was not
produced by
P. roqueforti
(Nielsen and
others 2006)
Tulum cheese P. roqueforti Penicillic acid, PR Thin-layer ND, ND, ND Erdogan and others
toxin, patulin, chromatography (2003)
roquefortine
Tulum cheese AFM1 LC-MS/MS 11 to 202 ng/kg Gürses and others
(2004)
Blue veined P. roqueforti Mycophenolic acid Thin-layer 250 to 5000 μg/kg Engel and others
chromatography (1982)
Blue mold P. roqueforti Roquefortine C LC-MS/MS 0.0008 to Kokkonen and others
Mycophenolic acid 0.012 μg/kg (2005a,2005b)
0.0003 μg/kg
Cheese P. camemberti Cyclopiazonic acid Thin-layer 0.5 to 1.5 μg/g Le Bars (1979)
chromatography
Gouda and Edam A. versicolor Sterigmatocystin Thin-layer 5 to 600 μg/kg Northolt and others
chromatography (1980)
Semhard cheese Penicillium sp. Patulin, ochratoxin High-performance 15 to 460 μg/kg and Pattono and others
liquid 1 to 262 μg/kg (2013)
chromatography
Turkish cheeses Aspergillus sp. Aflatoxin M1 ELISA 58 to 850 ng/kg Aydemir and others
(2010)
Italian white P. camemberti Cyclopiazonic acid High-performance 0.02 to 0.08 mg/kg Zambonin and others
cheeses liquid (2001)
chromatography

species encountered in cheese, the mycotoxin-producing ability mycotoxins. Analytical methods for identifying and quantifying
for some has already been well established, while for others mycotoxins in foods and feeds have widely been developed and
questions still remain. governmental agencies have establishe regulatory limits and guide-
lines for safe doses.
Mycotoxins in Cheese Various mycotoxins have been detected in cheese (Ta-
In the 1960s, the first studies related to mycotoxins in cheeses ble 1) and the most commonly identified are described below.
were published and led to the discovery of AFs. Other studies were Roquefortine C has been quantified in blue cheese (3.4 μg/g)
performed in the 1970s with special interest on the occurrence (Scott and others 1977), Manchego cheese (López-Diaz and oth-
of CPA in cheese. Since 2000 and the development of powerful ers 1996), and in commercial Finnish cheeses at levels reaching
analytical techniques such as LC-MS/MS, numerous studies have 12000 μg/kg (Kokkonen and others 2005a, 2005b). MPA has been
emerged on the subject. reported from different types of blue mold cheeses (1.5 mg/kg
As stated above, various mold species can be isolated from by Lafont and others1979 and 5 mg/kg by Engel and others
cheeses and correspond to ripening cultures or contaminating 1982) using thin-layer chromatography (TLC) and at much lower
molds (Taniwaki and others 2001) that can potentially produce levels (0.0003 μg/kg) in cheese using LC-MS-MS (Kokkonen

C 2014 Institute of Food Technologists® Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 439
Filamentous fungi and mycotoxins in cheese . . .
and others 2005a, b). Due to AFM1 occurrence in milk, this
mycotoxin can also be present in cheese. Various studies have re-
ported AFM1 in cheese (Gürses and others 2004; Aydemir and
others 2010). On the other hand, very few studies have described
AFG1 and AFB1 in cheese. In these cases, the mycotoxins were
directly added to cheese and their stability was followed over time.
Both AFB1 and AFG1 were stable in Swiss cheese (Lieu and
Bullerman 1977). Similar studies were also carried out by inocu-
lating A. flavus and Aspergillus parasiticus mycotoxinogenic strains
in cheese and both mycotoxins were again (Lie and Marth 1967).
Sterigmatocystin (STC) was detected and quantified in the surface
layer of A. versicolor contaminated hard cheeses at levels ranging
from 5 and 600 μg/kg (Northolt and others 1980). In white
cheese, CPA was detected from 0.5 to 1.5 μg/g (Le Bars 1979)
and from 0.02 to 0.08 mg/kg (Zambonin and others 2001). Stott
and Bullerman (1976) showed that when patulin was added to
cheddar cheese, it was not detected after storage by TLC. How-
ever, in semihard cheeses, patulin and ochratoxin were detected
at concentrations of 500 and 2620 μg/kg, respectively (Pattono
and others 2013). Since PR toxin was discovered (Wei and others
1973), no studies have described this mycotoxin in cheese as it is
unstable and leads to PR imine (Scott and Kanhere 1979; Siemens
and Zawitowski 1993).
Mycotoxin cheese contamination can occur indirectly via milk
contamination or directly by mycotoxin producing spoilage or
technological filamentous fungi (O’Brien and others 2004).
Concerning indirect contamination, the main concern is AFM1
in cheese. AFM1 in the final product is due to the addition of
this mycotoxin into the milk by dairy animals (Eaton and others
1994; Gallo and others 2008) that have fed on moldy AFB1-
contaminated feedstuffs. Indeed, AFB1 is converted into AFM1
by enzymes mainly present in the animal liver and then passed
into their urine and milk. Many studies have reported AFM1 in
various cheeses (Finoli and others 1983; Taniwaki and van Dender
1992; Gürses and others 2004; Kokkonen and others 2005b). Low
storage temperature of cheese can induce a decrease in AFM1 con-
centration. The indirect contamination of milk with other myco-
toxins, such as STC (van Egmond and Paulsch 1986), fumonisins
(Maragos and Richard 1994), or CPA (Dorner and others 1994),
has also been reported. Concerning this type of contamination
in cheese, it is much more complex because it depends on the
manufacturing processes used.
Direct mycotoxin contamination can be exogenous via the pres-
ence of contaminating molds during cheese-making or endoge-
nous by the presence of mycotoxin-producing commercial fungal
cultures.
Cheeses on which unintentional mold growth has occurred have
been reported to contain mycotoxins. However, mold growth
on the cheese surface does not automatically imply that myco-
toxins are present in cheese; moreover, mycotoxin production
can be quite different among mycotoxicogenic fungi (Moss 1991;
Sweeney and Dobson 1998).
In the case of direct contamination from an endogenous origin,
several cheeses that intentionally have added mold species during
cheese-making for appearance, texture and flavor development
can be at risk. Although many cheeses use molds for ripening,
the 2 main categories are: soft mold-ripened cheeses (such as
“Camembert”) associated with P. camemberti and blue-veined
cheeses (such as “Gorgonzola,” “St Nectaire,” and “Cantal”) as-
sociated with P. roqueforti. P. camemberti, and P. roqueforti have long
been used in the manufacture of mold-ripened cheeses consumed
440 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014
throughout the world. P. roqueforti is an essential component of the
microflora of many cheeses including Roquefort (France), Stil-
ton (U.K.), Tulum (Turkey), Gorgonzola (Italy), Cabrales (Spain),
Blauschimmelkase (Switzerland), and Danish Blue (Denmark)
(Bullerman 1981; O’Brien and others 2004), while P. camemberti is
associated with Camembert and Brie production. Both species are
known for their potential mycotoxin production; however, due
to cheese production practices and storage conditions, only low
to very low concentrations have been observed (Bullerman 1981;
Teuber and Engel 1983).
Overall, numerous mycotoxins can be found in cheeses at highly
variable concentrations depending on cheese origin.
Ecological role of mycotoxins in cheese
So far, the ecological roles of mycotoxins have not been com-
pletely elucidated but hypothesized functions in natural environ-
ments have been proposed. In the cheesemaking context, this role
has not been studied to our knowledge. Therefore, ecological role
hypotheses in this food matrix can only be proposed based on
studies performed in other environments.
Role in competition with other organisms. Mycotoxins are nat-
urally occurring molecules that are thought to confer a selective
advantage to the producer within complex ecosystems to com-
municate and compete with other organisms (Magan and Al-
dred 2007). Their widespread nature and the preservation of these
multigenic biosynthetic pathways suggest that secondary metabo-
lites are used for survival functions in organisms (Demain and
Fang 2000). The genes required for the biosynthesis of secondary
metabolites are clustered. As stated by Calvo and others (2002),
“logic suggests that their maintenance could only be selected for if the final
product conferred some advantage to the producing organism.” In this
context, mycotoxins have been proposed to act as chemical signals
for communication, a competitive weapon to defend the habitat,
or to inhibit the growth and reproduction of competitors of the
same trophic niche (Magan and Aldred 2007; Fox and Howlett
2008). The presence of competing microorganisms is essential to
mycotoxin production. these hypotheses are demonstrated by the
fact that serial transfers on culture media in laboratory conditions
(lack of exposure to competitive and natural stressed conditions)
result in the loss of AF production in strains of A. flavus (Horn and
Dorner 2001). Adverse conditions such as micro- and macronutri-
ent depletion and competition with other fungi have a stabilizing
effect in preventing AF production loss. In addition, early pro-
duction of mycotoxins could allow molds to rapidly colonize the
environment.
In the cheese ecological niche, both spoilage fungi and fungal
cheese cultures are in competition with other microorganisms in-
cluding bacteria and yeasts. One study showed bacteriostatic effect
of roquefortine on gram-positive bacteria, whereas gram-negative
bacteria were not influenced at all. This mycotoxin affected bac-
terial respiration (Kopp-Holtwiesche and Rehm 1990). However,
to our best knowledge, the effects of microbial competition on the
production of mycotoxins have not yet been studied in cheeses or
in cheese-mimicking models.
Role in fitness. Another possible role of mycotoxin produc-
tion is that it can be related to strain fitness. Mycotoxin pro-
duction could correspond to a mechanism for adaptation to the
environment. For instance, OTA biosynthesis by P. nordicum and
P. verrucosum increases their fitness under high NaCl concen-
tration conditions as exhibited in food products like dry-cured
meat or brined olives (Schmidt-Heydt and others 2012). OTA

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Filamentous fungi and mycotoxins in cheese . . .

Table 2–Examples of toxic effects of some mycotoxins produced by Penicillium species.

Mycotoxins Effects Models References

Mycophenolic acid LD50 (Letal Dose) rats 2500 mg/kg In vivo rats and mice Wilson (1971)
IP LD50 rats 500 mg/kg IV In vivo human Detroit 98 and Girardi Heart Engel and others (1982)
LD50 mice 700 mg/kg IP In vitro Salmonella sp. Umeda and others (1977);
LD50 mice 450 mg/kg IV In vitro human macrophage Webner and others (1978)
Mutagenic: chromosomes aberrations, Ames In vitro Caco cells Bentley (2000)
test Immunosuppressive Rasmussen and others (2011)
IC50 (inhibitory concentration) > 100 μg/mL
Roquefortine LD50 rats 1520 mg/kg In vivo rats and mice Ueno and others (1978)
LD50 mice 169 mg/kg M In vivo dogs Scott and others (1977)
LD50 mice 184 mg/kg F In vitro Salmonella sp. Puls and Ladyman (1988)
intoxications In vivo day-old cockerel Schoch and others (1984)
Ames test-neurotoxic In vitro Caco cells Wagener and others (1980)
IC50 48 μg/mL Rasmussen and others (2011)
PR toxin LD50 mice 5.8 mg/kg IP In vivo mice and rats Chen and others (1982)
LD50 rat 11.6 mg/kg IP In vitro E. coli Moule and others (1976)
LD50 rat 8.2 mg/kg IV In vitro rat liver mitochondria Wei and others (1984)
Target organs: capillary permeability (heart, In vitro Salmonella sp. Ueno and others (1978)
liver, lungs, kidneys damages) In vitro S. cerevisiae Wei and others (1979)
inhibits RNA and protein synthesis inhibits
DNA polymerase
mitochondrial respiration
mutagenic (Ames+), carcinogenic
Penicillic acid Synergistic with OTA In vivo pig Stoev and others (2001)

contains a chloride atom within its molecule and the constant
OTA biosynthesis ensures permanent excretion of chlorine out of
the cell for Penicillia (Schmidt-Heydt and others 2012). The more
constantly a strain produces OTA, the higher its growth rate will
be under high NaCl conditions. Consequently, a strain competes
better on a salt-rich habitat in comparison to a strain that produces
low amounts of OTA (Schmidt-Heydt and others 2013). Cheese
contains a fair amount of NaCl and it is also likely that the NaCl
level may influence OTA production in cheeses.
Role in detoxification. Finally, mycotoxin production could
correspond to a waste elimination process. Indeed, mycotoxins
could contribute to cell detoxification from the accumulation
of reactive oxygen species (Reverberi and others 2010; Sanzani
and others 2012). Fungi are aerobic organisms producing reac-
tive oxygen species in their environment that can damage cell
membranes and metabolic pathways. Generally, a cell can toler-
ate oxidative stress by increasing antioxidant synthesis and also
secondary metabolite production, such as mycotoxins, which are
under the control of reactive oxygen species (Reverberi and oth-
ers 2010). In cheese, most LAB are able to produce hydrogen
peroxide (H2 O2 ) in the presence of oxygen. Hydrogen perox-
ide can then be converted to hypothiocyanate (HOSCN) via the
reaction of hydrogen peroxide and thiocyanate through catalysis
by lactoperoxidase that are both naturally present in milk. These
reactive oxygen species, besides the fact that they have known
antifungal activities (Schnürer and Magnusson 2005), may also,
in turn, affect mycotoxin production in cheese-related fungi. We
could also speculate that the production of MPA may be a route
for detoxifying acetate, another well-known fungal inhibitor pro-
duced, for example, by nonstarter heterofermentative LAB such
as Lactobacillus spp., however, no scientific evidence yet exists to
support that these hypotheses are true.
Mycotoxin production and their biological roles must be con-
sidered in an ecological context, which is not easy to elucidate in
laboratory conditions. In fact, mycotoxigenic molds do not occur
alone, they form part of an ecological niche in which diverse com-
munities of microorganisms compete. A better understanding of
the regulation and role of mycotoxin production during cheese-
making could help to develop approaches to control mycotoxin
contamination in cheeses.
Nature and toxicity
The usual routes for mycotoxin exposure are ingestion of
mycotoxin-contaminated foods and feeds (Hayes 1980; Sweeney
and Dobson 1998; Hussein and Brasel 2001; Bennett and Klich
2003), physical contact (CAST 2003), and inhalation (Bunger and
others 2004) (Table 2).
Mycotoxins can cause either acute or chronic diseases in animals
and humans (Stoloff 1977; Pitt 2000) depending on the dose,
ingestion duration, and animal species.
Mycotoxins present in cheese due to fungal contamination.
Aflatoxins. AFs derive from polyketide metabolism and con-
sist of at least 16 structurally related furanocoumarins of which
AFB1, AFB2, AFG1, and AFG2 are the most abundant (Huff-
mann and others 2010). AFs are considered to be among the most
significant classes of known mycotoxins in food and feed. They
are produced by certain Aspergillus species. Highest AF levels are
produced as a result of inadequate postharvest storage when high-
moisture content and warm temperatures facilitate rapid mold
growth. Therefore, the presence of AFs in animal feedstuffs (ce-
reals, forages, and so on) (Garon and others 2006) can be an
important contamination source for the dairy industry. As stated
above, various studies have reported AFM1 in cheese. Concern-
ing other AFs, AFG1, and AFB1 have been reported in cheese,
but only after spiking samples with the mycotoxins or mycotox-
inogenic strains (Gürses and others 2004; Aydemir and others
2010).
Lethal dose (LD50) values range from 0.5 to 10 mg/kg according
to the considered AF. Animal species respond differently in regard
to their susceptibility to chronic and acute AF toxicity. During
aflatoxicosis, the liver is the primarily affected organ, leading to
bile duct proliferation, centrilobular necrosis, fatty acid infiltration,
and hepatic lesions (Heinonen and others 1996; Bhatnagar and
others 2002), which ultimately lead to liver cancer (Chao and
others 1991; Sweeney and Dobson 1998). Other organs such as
the kidney, lung, heart, gizzard, or spleen may also be affected
(Yang and others 2012).
AFs induce DNA damage, negatively affect damage repair, and
alter DNA base compositions of genes. AF is associated with both
toxicity and carcinogenicity in human and animal populations.
Within the AF family, IARC (International Agency for Research


C 2014 Institute of Food Technologists® Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 441
Filamentous fungi and mycotoxins in cheese . . .
on Cancer 1993) has classified AFB1, as a group class 1 proven to
cause cancer in human.
Although many different cheese types can contain AFM1, it
is almost invariably at such low levels that no danger to human
health exists (Blanco and others 1988). But recent studies showed
that high AF levels up to 850 ng/kg in regional Turkish cheese
(Aydemir and others 2010). Therefore, the health risk should not
be underestimated.
Finally, due to AFM1 toxicity (classified by the IARC 2002,
as class 2B, possible human carcinogen), many countries tightly
monitor AFM1 levels in milk and milk products and, as stated
above, the European Union has established a 0.05 μg/kg maximal
limit (EFSA 2004).
Sterigmatocystin. STC is a xanthone and is structurally similar
to AF (Hamasaki and Hatsuda 1977); however, it is less toxic
(Sweeney and Dobson 1998). This mycotoxin is produced by a
few Aspergillus species (Cole and Cox 1981; Veršilovskis and De
Saeger 2010).
STC has indeed been detected in A. versicolor-contaminated
cheeses (Scott 1989). Northolt and others (1980) reported STC
occurrence during cheese (Gouda and Edam) ripening in ware-
houses in the Netherlands. STC was stable in contaminated cheeses
for a 3-mo period at various temperatures (−18 to +16 °C) (van
Egmond 1983). However, low temperatures (5 to 7 °C) used dur-
ing ripening and storage should prevent A. versicolor growth and
STC production. At these temperatures, molds that develop on
cheese rather correspond to Penicillium species, which could not
produce AFs or STC (Bullerman 1981). Nevertheless, STC was
analyzed in cheese samples, produced in Latvia (8) and Belgium
(13). Two cheeses (9.5%) were positive for STC with concentra-
tions of 1.23 and 0.52 g/kg, respectively. Five (24%) samples con-
tained STC above the limit of detection (0.03 g/kg) (Veršilovskis
and others 2009).
STC is activated to sterigmatocystin-1,2-epoxide by liver en-
zymes, which binds covalently to DNA and induces various tu-
mors (Baertschi and others 1989; Raney and others 1992). STC
produces higher cytotoxicity than AFB1 (IC50 = 10 μM) in A-
549 cultures (lung cell line) (Palanee and others 2001). STC is
carcinogenic in animal models and possibly carcinogenic to hu-
mans (IARC 1976).
Citrinin. Citrinin can be produced by a dozen Penicillium
species including a few P. camemberti strains and several Aspergillus
species (Manabe 2001). P. citrinum, P. viridicatum, P. expansum, P. ver-
rucosum, A. carneus, A. niveus, and A. terreus have all been reported
to be the most important citrinin-producing fungi in nature, but
other species such as Aspergillus candidus, have also been found to
produce citrinin under laboratory conditions (Raper and Thom
1949; Frisvad and others 2006). In cheese, P. citrinum and P. verruco-
sum only are known to produce this mycotoxin. Citrinin appeared
to be very stable in cheeses, including goat cheese, Saint Marcellin,
Soignon (Bailly and others 2002). This mycotoxin is frequently as-
sociated with the presence of OTA both produced by P. verrucosum
(Cole and Cox 1981; Ostry and others 2013).
Citrinin was shown to be nephrotoxic on all performed animal
experiments (Kitchen and others 1977). LD50 is 57 mg/kg for
ducks, 95 mg/kg for chickens, and 134 mg/kg for rabbits. EFSA
(2012) estimated based on a 90-d toxicity study in rats, a no-
observed-adverse-effect level (NOAEL) of 20 μg citrinin/kg b.w.
per day for nephrotoxicity.
Citrinin can act synergistically with OTA to depress RNA syn-
thesis in kidneys of mice (Sansing and others 1976). Citrinin
also induces a loss of selective membrane permeability and cell
442 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014
death (possibly apoptotic) (Paterson and Lima 2010). Citrinin has
a high IC50 in vitro on Caco-2 cells (83 μg/mL) but can exhibit
nephrotoxic effects in mammals (Rasmussen and others 2011).
Citrinin exhibits immunotoxic effects, inducing lymphocyte pro-
liferation inhibition in pigs. An immunosuppressive effect is esti-
mated at IC50 equal to 10 mg/L (Wichmann and others 2002).
On embryonic stem cells, citrinin induced apoptosis via ROS and
mitochondria-dependent pathway (Chan 2007).
Ochratoxin. OTA is produced by several Aspergillus (Benford
and others 2007) and Penicillium species. In temperate and cold cli-
mates, P. verrucosum is the major producer of OTA in cereals. P. ver-
rucosum is recovered mainly from plant-derived material; P. nordicum
is generally recovered from meat and cheese products (Cabanes and
others 2001). OTA has been detected in a wide range of foods
and feed, including cereals, bread and beer (Visconti and others
2010) as well as meat and cheese products (Gareis and Scheuer
2000). OTA was originally discovered as an Aspergillus ochraceus
metabolite during a large metabolite screening procedure (Van der
Merwe and others 2000) and shortly these after recognized as a
potent nephrotoxin, in particular in swine and poultry. Among the
numerous ochratoxin analogs (at least 20), OTA is produced at the
highest levels and is the most toxic. Although OTA transfer into
milk has been demonstrated in other animal species, in ruminants,
the resident rumen microflora decreases bioavailability through
hydrolysis of OTA to OTα (Skaug 1965). Nevertheless, OTA
has been detected in different commercial samples of blue-veined
cheeses (Gorgonzola and Roquefort), although only at very low
levels (Dall’Asta and others 1999). The same authors showed that
32.6% of blue cheeses inoculated with only P. roqueforti (non-OTA
producers) were actually contaminated with OTA (Dall’Asta and
others 2008). OTA levels detected in different cheese portions
demonstrated a nonhomogeneous contamination highly associ-
ated with the blue–green veins of the cheese where fungal growth
occurs. Moreover, the observed levels increased during storage,
indicating that contamination was not derived from contaminated
milk. This implied that OTA-producing fungal strains may have
contaminated the cheese during the manufacturing process.
Ochratoxins have been known for some time to be a ma-
jor etiological agent in endemic nephropathy (Radic and others
2008) and, by analogy, they have been considered to be associated
with the chronic progressive kidney disease in humans known as
Balkan endemic nephropathy (BEN) and urinary tract tumors
(Castegnaro and others 1997; Tanchev and Dorossiev 1991;
O’Brien and Dietrich 1991). Initiation of carcinogenic process
is the covalent binding of OTA to DNA, forming DNA adducts
(Lebrun and others 2005). OTA toxicity involves mitochondrial
respiratory function, OTA interferes in modulating CYP 450 en-
zymes, COX, and lipox expression (Pfohl-Leszkowicz and Man-
derville 2006). OTA induced Ca2+ increase into the cells, tRNA-
synthase inhibition, accompanied by reduced protein synthesis and
enhanced lipid peroxidation via the generation of free radicals
(Höhler 2007). Intake of OTA from cheese seems to be of limited
importance for the general population in comparison to potential
OTA intake from cereals and their derived products.
Mycotoxins potentially produced by the commercial cheese mold
P. roqueforti.
Roquefortine C. Roquefortine C is the most common metabolite
produced by Penicillium genus members (25/58 species) (Frisvad
and others 1998). Moreover, according to Scott (2004) and Vi-
nokurova and others (1981), within the P. roqueforti species, vir-
tually all studied strains produce roquefortine C and its precursor
roquefortine D.

C 2014 Institute of Food Technologists®
Filamentous fungi and mycotoxins in cheese . . .

Roquefortine C was previously incriminated in intoxication species (Pitt and others 1986). Spahr and others (2000) considered
cases in cattle in Germany (Häggblom 2001; Auerbach and oth- that CPA is one of the few mycotoxins that can be transferred to
ers 1990). To date, there are no toxicity studies performed on milk. CPA is a calcium-dependent ATPase inhibitor and induces
laboratory animals that meet good laboratory practice standards. ion transport alteration across cell membranes (Riley and Goeger
Intraperitoneal (IP) lethal dose 50 (LD50) were 169 mg/kg and 1992; Burdock and Flamm 2000). Clinical intoxication signs in-
184 mg/kg in male and female mice, respectively (Arnold and clude weight loss, anorexia, diarrhea, dehydration, pyrexia, ataxia,
others 1998). Wagener and others (1987) reported paralytic ac- immobility, and death in dogs, rats, pigs, sheep, and chickens (Bry-
tivity in day-old cockerels exposed to roquefortine C. In vitro, den 1991). The possible role of CPA in aflatoxicosis cases should
roquefortine C exhibits an inhibitory concentration 50 (IC50 ) of be explored as it could have a synergistic effect. Target organs
48 μg/mL on Caco-2 cells (2011Rasmussen and others 1980). are kidneys and gut tract in mammals. In acute toxicity, LD50 is
However, no recent data are available to estimate human exposure 64 mg/kg in female mice and 12 mg/kg in chickens. In humans,
(Table 2). To date, roquefortine C has never shown any mutagenic CPA is suspected to be responsible for acute mycotoxicosis (named
activity by the Ames test (Schoch and others 1984). Roquefortine “kodua”) inducing nerve troubles.
C is known to be neurotoxic (Frisvad and others 2007). There are There is limited information and only few data concerning my-
a number of dog toxicosis cases linked to roquefortine C (Puls and cotoxin toxicities associated with P. roqueforti and P. camemberti.
Ladyman 1988), but penitrems or other compounds might have Not enough is currently known about the actual toxicity of my-
also been present. cotoxins found in cheeses and the toxicity of metabolites that can
Mycophenolic acid. Few studies reporting the presence of MPA co-occur with mycotoxins.
produced by P. roqueforti in cheeses have been carried out on
blue cheeses (Lafont and others 1979; Engel and others 1982;
López-Diaz and others 1996). MPA is a phthalide and although it Methods for qualitative and quantitative analysis (Table 1)
is a mycotoxin with potential toxicological implications, it is also Due to mycotoxin toxicity, their frequent occurrence in food-
a very important drug with several potential applications (Bentley stuffs, and driven by regulatory authorities worldwide, there is a
2000). It is presently used as an immunosuppressant in kidney, distinct need for highly selective and accurate methods to iden-
heart, and liver transplant patients to avoid organ rejection. Toxi- tify and quantify mycotoxins in diverse agricultural and biological
city for mammals appears to be low: LD50 in rats is 2500 mg/kg foods and feeds. For example, the maximum AF levels for animal
(oral pathway) and 500 mg/kg by intravenous (IV) pathway in feed are 50 and 20 ppb in the United States and Europe, respec-
mice the LD50 is 700 mg/kg and 450 mg/kg IV (Wilson 1971). tively. There are no published guidelines for mycotoxin analysis.
Chronic tests of 80 and 320 mg/kg daily oral doses over 1 y did Numerous methods have been developed to analyze the large
not cause apparent toxicity signs in rabbits (Adams and others structural diversity of mycotoxins occurring in different matrices
1975). It is not unlikely that MPA could lower the immune sys- (Zöllner and Mayer-Helm 2006; Malik and others 2010).
tem if ingested often enough thereby paving the way for bacterial Chromatography. Concerning cheese, classical analytical meth-
infections (Bentley 2000; Frisvad and others 2007). Mutagenic ods for mycotoxin detection and/or quantification include TLC,
activity was evaluated and showed that this mycotoxin was able to high-performance liquid chromatography (HPLC), gas chro-
induce chromosome aberration (Umeda and others 1977) but was matography (GC), and mass spectrometry (MS) coupled with
negative for the Ames test (Webner and others 1978). chromatography. One of the disadvantages for GC analyses for
Concerning MPA, with the exception of immunotoxicological mycotoxin detection is the necessity to derivatize samples, a time-
effects, toxicological data are scarce. consuming and prone to error step; as a result, GC methods are less
Other mycotoxins. In regards to other mycotoxins produced by frequently used. In recent years, most of these methods have been
P. roqueforti in cheese, only few toxicological data are available. coupled with immunoaffinity techniques to simplify extraction
Isofumigaclavines (A and B), festuclavine, and agroclavine are and improve mycotoxin recovery and measurement from food-
alkaloids potentially produced by P. roqueforti. They belong to the stuffs (Bony 2000; Chu 2000; Miraglia and Brera 2000; Stroka
ergot alkaloid mycotoxin family (Bräse and others 2009). Isofumi- and others 2000a, 2000b; Anon 2001; Josephs and others 2001;
gaclavines A and B have been detected at low levels in commercial Krska and Josephs 2001; Turner and others 2009).
blue cheeses (O’Connor and O’Brien 2000). Isofumigaclavine B As cheese has a high fat content, hexane was simultaneously used
corresponds to the isofumigaclavine A hydrolysis product. These to remove lipid compounds for the partitioning and extraction
toxins were reported by Ohmomo and others (1975, 1977) and by steps. However, some lipids persist during extraction; therefore,
Kozlovskii and others (1979). Agroclavine increased natural killer pure ACN, or methanol is also used to dissolve the sample in the
(NK) cell activity under nonstress conditions in vivo and in vitro, final step.
thus exhibiting an immunostimulatory effect (Starec and others HPLC is widely used to quantify different mycotoxins in
2001). cheeses, including AFM1 (Kamkar 2008). HPLC reference meth-
PR toxin (Table 2), another known mycotoxin produced by ods have been developed for most of the major mycotoxins and are
P. roqueforti, is unstable in the cheese environment and is con- quite sensitive with reasonably low detection levels. For example,
verted to PR imine, an unstable and less toxic molecule. PR toxin patulin and ochratoxin are detected in traditional semihard cheeses
can also be converted to PR amide in the presence of basic and at levels ranging from 15 to 460 and 1 to 262 μg/kg, respectively
neutral amino acids (Chang and others 1993). Other secondary (Pattono and others 2013).
metabolites related to PR toxin synthesis by P. roqueforti, such as LC/MS or GC/MS have excellent detection sensitivity and
eremofortins A, B, C, and D, have also been observed. selectivity. The better sensitivity of new MS instruments, such
Mycotoxins potentially produced by the commercial cheese mold as those equipped with ion trap analyzer, in combination with
P. camemberti. better GC columns, can lead to use the GC/MS technique to
Cyclopiazonic acid. CPA, an indole tetramic acid, is produced significantly low patulin levels for example. However, the neces-
by P. cyclopium, some P. camemberti strains, and several Aspergillus sity of derivatization makes exact quantification more adapted for


C 2014 Institute of Food Technologists® Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 443
Filamentous fungi and mycotoxins in cheese . . .

LC rather than GC/MS, unless a stable isotope-labeled patulin is a single genetic locus (Hoffmeister and Keller 2007). This fea-
available as internal standard. ture, combined with the fact that some of the most important
LC/MS is an analytical technique independent of molecular mycotoxins are synthesized by polyketide synthases (PKSs) (AFs,
weight so that mycotoxins can be detected and quantified in com- fumonisins, ochratoxins, and zearalenone), nonribosomal peptide
plex matrices like cheeses. However, efforts must be made to de- synthetases (NRPS) (gliotoxin, enniatin, and HC-toxin), or PKS-
velop a wide range of protocols taking into account analyte polarity NRPS hybrid proteins (CPA), allowed to identify biosynthetic
diversities and different ionization capacities. Liquid chromatog- pathways and the involved genes in various species (Figure 1).
raphy tandem mass spectrometry (LC/MS/MS) is a suitable tech-
nique as it can be used to simultaneously analyze all mycotoxins
produced by 1 fungus (Xu and others 2006). The HPLC/MS/MS
Biosynthetic pathways in undesirable species
For cheese-contaminating fungi, the pathways associated with
performance for cheese samples was defined by recovery, repeata-
mycotoxin production have been studied in depth from a genetic
bility, and limit of quantifications (LOQs). No regulatory limits
point of view.
exist for cheese, so LOQs cannot be evaluated.
Aflatoxins/sterigmatocystin. The biosynthetic genes and reg-
Recently, Kokkonen and others (2005b) proposed an easy
ulation involved in AF and STC synthesis have been extensively
LC/MS/MS method for quantifying several mycotoxins in blue
documented in Aspergillus species (Keller and Hohn 1997; Minto
and white mold cheeses; the proposed method detected 9 myco-
and Townsend 1997; Payne and Brown 1998; Brown and others
toxins produced by Aspergillus and Penicillium species. Mycotoxins
1999; Yu and others 2004; Yabe and Nakajima 2004; Georgianna
(AFs, OTA, MPA, and roquefortin C) were quantified from cheese
and Payne 2009; Huffman and others 2010) including A. flavus
samples at low levels ranging from 0.6 to 5.0 μg/kg.
(pathway discovered in this AF producing cheese contaminant)
Liquid chromatography tandem mass spectrometry
(Barrios and others 1998; Mogensen and others 2010) and A.
(LC/MS/MS) analytical methods are usually used to con-
parasiticus (Yu and others 2004). In regards to cheese contamina-
firm data since they require expensive instrumentation and
tion and as previously stated, detection of these mycotoxins in
highly trained staff. Recently, the natural occurrence of masked
cheeses is typically due to indirect cheese contamination via feed-
mycotoxins (conjugated toxin) has been reported, and their
stuffs (this is the case for AFs and AFM1 produced by Aspergillus
detection will require even more, selective, and sensitive methods
species on feedstuffs that are further detected in milk and cheese)
(Berthiller and others 2005).
or postcontamination (mostly relates to STC in cheese due to A.
Bioassay techniques. Bioassays have become increasingly useful
versicolor mold growth on cheese surfaces (Northolt and others
for mycotoxin detection (Watson and Lindsay 1982; Yates 1986)
1980). Among the studied chesse-related and noncheese-related
as a rapid screening procedure before chemical analysis. Screening
species, gene organization can differ and may indicate different
assays used are in majority immunochemical methods including
evolutionary histories; this is the case of A. nidulans and A. flavus
enzyme-linked immunosorbent assays (ELISAs), lateral flow de-
(Cary and others 2009). For cheese-related Aspergillus spp., the
vices (LFDs), dipstick tests, fluorescence polarization immunoas-
AF biosynthetic pathway was shown to contain 25 genes clustered
say (FPIA), immunofiltration assays, and more recently, biosensor
within a 70-kb region and involves at least 23 enzymatic reac-
assays.
tions (Yu and others 2004; Huffman and others 2010). The starter
Immunochemical methods can also be considered as convenient
units correspond to acetate and malonyl-CoA that are converted
and sensitive alternatives for detecting various mycotoxins in
sequentially to hexanoate by a fatty acid synthase (FAS) and to nor-
cheese (Chu 1991; Mak and others 2010; Mohajeri and others
solorinic acid (common to both AF and STC biosynthesis) by a
2013). For example, both direct and indirect ELISA strategies have
PKS (Crawford and others 2006, 2008a, 2008b, 2009). Expression
clear advantages (cost and time efficiency) and only few limitations
of all pathway genes, except for aflS (formerly known as aflJ and
including cross-reactivity and immunoreactivity of the primary
also involved in AF regulation), has been shown to be positively
antibody resulting in nonspecific signals (Turner and others 2009).
regulated by a transcription factor, aflR. It is known that STC
There are a lot of commercially available bioassay kits. For example,
is the final metabolic product of the biosynthetic pathway in A.
available ELISA kits include, Agraquant (RomerLabs Diagnotica
nidulans, while this mycotoxin acts as a precurseur for AF biosyn-
GmbH, Tulln, Austria) and Veratox (Neogen Corporation, Lesher
thesis in other Aspergillus species including A. flavus and A. par-
Place Lansing, Mich., U.S.A), the LFD kit “RIDAQUICK” (R-
asiticus (Yu and others 2004; Huffman and others 2010). These
Biopharm AG, Darmstadt, Germany) are avaible in Europe but
biosynthetic pathways have been extensively reviewed by Yu and
no bioassay kit is specific to cheese samples. Additionally, not all
others (2004) and Huffman and others (2010), and the detailed
mycotoxins (including patulin) can be measured by these methods.
gene cluster is provided in Figure 1 for A. flavus, a species in-
As presented, numerous techniques are currently available to
volved in indirect mycotoxin contamination in milk and cheese.
analyze and detect a wide spectrum of mycotoxins. However,
Ochratoxin. A pathway for OTA biosynthesis was recently re-
powerful methods still need to be developed to detect and quantify
viewed by Huffman and others (2010) and includes a polyketide
multiple toxins from a single matrix. Due to the high complexity
synthase for PK dihydroisocoumarin synthesis, a methyltransferase,
of the cheese matrix, LC/MS/MS currently seems to be the most
a P450-type oxidation enzyme for carboxyl group formation at
sensitive method to detect and quantify mycotoxins in cheese.
C7, a NRPS to catalyze ligation between phenylalanine and PK
and a halogenase to incorporate the chlorine atom. To date, char-
Mycotoxin Biosynthetic Pathways acterization of part of the OTA biosynthetic gene cluster has been
As stated above, mycotoxins correspond to a structurally di- described in the cheese contaminating species Penicillium nordicum
verse group varying from simple C-compounds to more complex (Karolewiez and Geisen 2005; Geisen and others 2006). Geisen
substances (Bräse and others 2009). These compounds are part and others (2006) sequenced a 10-kb-length fragment contain-
of fungal secondary metabolism, and interestingly, unlike primary ing 3 genes corresponding to a partial PKS (otapksPN), a com-
metabolism-associated genes, a large number of the mycotoxin plete NRPS homolog (npsPN) and a complete alkaline serine
production-associated genes have been shown to be clustered on protease homolog (aspPN). Moreover, O’Callaghan and others

444 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014 
C 2014 Institute of Food Technologists®
Filamentous fungi and mycotoxins in cheese . . .

Sterigmatocystin / Aflatoxin– A. flavus

O OH
aflY aflX aflW aflV aflK aflQ aflP aflO aflI aflL aflG aflN aflM aflE aflJ aflH aflS aflR
O O

O O

aflB aflA aflD aflC aflT aflU aflF O O

O

O
O OCH3

Citrinin – M. aurantiacus
ctnD cdnE orf6 orf1 ctnA pksCT orf5 ctnF orf7 ctnR orf8 ctnG ctnH
O

O OH

OH O
Ochratoxin – P. nordicum
O OH
asp npsPN otapksPN O OH O

NH O

Cl
Roquefortine C - P. chrysogenum O
rpt gmt P450 N-hydrolase hydrolase rds NH
N
N
O
H N
N
Mycophenolic acid - P. brevicompactum H

mpaH mpaG mpaF mpaE mpaD mpaC mpaB mpA OH O

HO
O
O
O

PR toxin - P. roqueforti O
ari 1 O
O O
O
O

Cyclopiazonic acid – A. flavus O OH

N
cpaO cpaD cpaS H
H O
H

N
H

Figure 1–Known fungal gene clusters associated with production of mycotoxins encountered in cheese. Based on the following Genebank accession
numbers: sterigmatocystin/aflatoxin (NW_002477243), citrinin (AB243687), ochratoxin (AY557343), roquefortine C (AM920436), mycophenolic
acid (HQ731031), PR toxin (L05193), and cyclopiazonic acid (JN712210).

(2013) recently described a 3-gene cluster encoding an oxidore-
ductase (OtaE), a PKS (OtapksPV), and a transporter protein
(OtaT) involved in OTA biosynthesis in P. verrucosum, another
cheese contaminating species. Other Penicillium species carry in-
active homologs for these genes (P. nalgiovense, also isolated from
cheese) or do not carry these genes. However, detailed genetic
and biochemical studies are still required to elucidate the molec-
ular mechanisms and confirm the proposed OTA biosynthetic
pathway.
Citrinin. Citrinin is known to be produced by some
cheese contaminating Penicillium species including P. citrinum,
P. verrucosum, and P. expansum (Ei-Banna and others 1987; Ku-
rata 1990; Cabanes and others 2007). However, to this date, cit-
rinin biosynthetic pathway has not been characterized in these
species but can be extrapolated from putative citrinin biosynthetic
16-gene cluster recently described in Monascus purpureus (Shimizu
and others 2005, 2007) and M. aurantiacus (Li and others 2012).
In these species, the pathway genes were identified to be part of
a putative 43-kb biosynthetic gene cluster including a polyketide
synthase gene (pksCT), a transcriptional activator gene (ctnA), a
membrane transporter gene (orf5), a fatty acyl-CoA synthetase
(ctnI), an oxygenase (orf3), 3 genes for post-PKS-modifying en-
zymes, oxidoreductase genes (ctnD, orf4), and 3 dehydrogenases
(ctnE, orf1, ctnH) among others (Shimizu and others 2005; Li and
others 2012). Biosynthesis of citrinin, originating from a polyke-
tide, often co-occurs with OTA. Further studies are still required
to fully understand the regulation of citrinin biosynthesis.
Biosynthetic pathways in cheese ripening species
Concerning the potentially mycotoxigenic fungal species used as
ripening cultures for cheese, P. roqueforti and P. camemberti, biosyn-
thethic pathway-associated genes have not been described to this
date, with the exception of the PKS involved in PR toxin for-
mation. However, pathways have been identified in other mold
species. These studies can be used as a base for studying mycotoxin-
related genes in fungi used as ripening cultures in cheesemaking.
Roquefortine C. Studies have shown that roquefortine C is
an intermediate of the oxaline and epi-neoxaline pathway in
P. glandicola (Reshetilova and others 1995) and P. tulipae (Overy
and others 2005), respectively. In 2011, a biosynthesis pathway
was proposed in P. chrysogenum (Garcıá-Estrada and others 2011).
In this study, by using gene silencing, the authors indicated that a
single-gene cluster containing 6 genes involved in the biosynthe-
sis and secretion of 2 mycotoxins: roquefortine C and meleagrin.


C 2014 Institute of Food Technologists® Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 445
Filamentous fungi and mycotoxins in cheese . . .
The cluster organization showed the presence of genes encod-
ing a nonribosomal cyclodipeptide synthetase, a prenyltransferase,
and an N-hydroxylase leading from histidine and tryptophan to
roquefortine D. Under the action of a monooxygenase, the latter
substance is metabolized into roquefortine C. The production of
meleagrin is obtained via the actions of 3 enzymes leading se-
quentially to glandicoline A and glandicoline B before meleagrin.
More recently, 2 publications refined the biosynthetic pathway and
reassigned ascribed synthetases. According to Hazrat and others
(2013), the NRPS RoqA is indeed responsible for the production
of histidinyltryptophanyldiketopiperazine (HTD). The molecule
can be transformed by the sequential action of RoqR followed
by RoqD, or conversely by the action of RoqR and then RoqD,
to lead to roquefortine C. This is done without the action of
RoqM contrary to what was proposed by Garcıá-Estrada and oth-
ers (2011). The same research group showed further branching in
the metabolic pathway by the characterization of roquefortine F
and neoxaline as well as the identification of Roquefortine L and
its degradation products (Ries and others 2013). Noteworthy, in
P. roqueforti and in the cheese context, only roquefortine C has
been observed, thus suggesting that a part of the pathway leading
from roquefortin C to meleagrin might either be absent or inactive
in this species.
Mycophenolic acid. As described by Birch and others (1957),
MPA consists of an acetate-derived phthalide nucleus and a
terpene-derived side chain indicating the involvement of polyke-
tide and isoprenoid pathways. Proposed biosynthetic pathways
(Bentley 2000; Regueira and others 2011) indicate that acetyl-
CoA, malonyl-CoA (x3), and S-adenosyl methionine (SAM)
would form 5-methylorsellinic acid (5-MOA) through the action
of a polyketide synthase. Following phthalide formation, a reac-
tion with farnesyl diphosphate yields 6-farnesyl-5, 7-dihydroxy-
4-methylphthalide. Degradation of the side chain via oxidative
cleavage leads to demethylmycophenolic acid (DMPA), which is
finally methylated by a SAM: DMPA O-methyltransferase to yield
MPA. This last step was confirmed by the purification and charac-
terization of SAM: DMPA O-methyltransferase in P. stoloniferum.
Regueira and others (2011) identified and characterized the MPA
biosynthetic gene cluster (8 putative genes) in P. brevicompactum.
Gene cluster determination was performed by detection of a re-
sistance gene encoding an inosinate dehydrogenase (IMPDH) and
deletion of the mpaC gene encoding a PKS, which abolished the
production of metabolites (including MPA), associated with MPA
biosynthesis. In the described cluster, the mpaG gene encodes the
SAM: DMPA O-methyltransferase. Recently, Hansen and others
(2012) showed through the creation of a chimeric MpaDE pro-
tein, consisting of a cytochrome P450 (encoded by the mpaD
gene) and a hydrolase (encoded by the mpaE gene), that the step
following 5-MOA production in the pathway could be carried
out by a natural-fusion MpaDE enzyme.
PR toxin. PR toxin metabolic pathways have been proposed
in P. roqueforti (Moreau and others 1980; Chalmers and others
1981; Jelén 2002). In the pathway, Chalmers and others (1981)
proposed that the pathway precursor, acetate, is sequentially con-
verted to mevalonate, dimethylallyl pyrophosphate (DMAPP), and
farnesyl pyrophosphate (FPP). Cyclizations by the aristolochene
synthase lead to aristolochene, the precursor for eremophilane-
type sesquiterpenes, with germacrene A as an intermediate, as
demonstrated by Calvert and others (2002). PR toxin arises from
aristolochene via a synthetic pathway including eremefortin B, A,
and C. Eremefortin C is finally converted to PR toxin. The ac-
etal form of eremofortin C leads to eremofortin D (Moreau and
446 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014
others 1980). Recently, Brock and Dickschat (2013) used a com-
bination of CLSA/GC-MS and 13 C NMR to obtain an in-depth
view of the biochemical pathway and described several unknown
sesquiterpenes leading from FPP and aristolochene as well as side
products. PR toxin can be degraded in PR imide, PR amide
(eremofortin E) (Chang and others 1993), and PR acid (Chang
and others 1996). Concerning the genes and enzymes associated
with this pathway, until recently, the genes involved in this path-
way had not been described, with the exception of aristolochene
synthase, a sesquiterpene cyclase, implicated in PR toxin biosyn-
thesis in P. roqueforti (Proctor and Hohn 1993). The 1129-bp-long
Ari1 gene, exhibiting 2 introns, encodes a 342-amino acid protein
with a calculated molecular weight of 39200 (Proctor and Hohn
1993). The protein was purified by Hohn and Plattner (1989) and
was shown to be Mg2+ -dependent. The crystal structure (2.5-Å
resolution) was determined by Caruthers and others (2000) and
confirmed that the enzyme is responsible for the cyclization of
the FPP, to form aristolochene. The enzyme eremefortine C, re-
sponsible for the conversion of eremefortin C to PR toxin, was
studied by Chang and others (1985). Recently, Hidalgo and others
(2014) cloned and sequenced a 4-gene cluster from P. roqueforti.
The cluster included the ari1 gene and silencing of the other genes
(encoding for putative oxidoreductases and an alcohol dehydroge-
nase) demonstrated their involvement in the PR synthetic pathway.
Interestingly, silencing of the PR toxin production caused a large
increase in MPA synthesis suggesting a cross talk between these 2
mycotoxin pathways.
Cyclopiazonic acid. CPA, produced by P. camemberti in cheese,
derives from tryptophan, acetyl CoA, malonyl CoA, and DMAPP,
as a prenyl donor, via the formation of 2 intermediates,
cycloacetoacetyl L-tryptophan (cAATrp) and β-CPA (Holzapfel
and Wilkins 1971). As stated above and as for the other mycotoxins
described in this section, to date no complete biosynthetic cluster
associated to cheese ripening cultures has been elucidated. How-
ever, for CPA, gene clusters were described in A. flavus (cheese-
contaminating species) and A. oryzae and can be used as a base
to elucidate the corresponding pathway in P. camemberti. In A.
flavus, the CPA biosynthetic pathway consisted of 3 genes (cpaS,
cpaD, and cpaO) and was situated next to the AF biosynthesis
gene cluster (Chang and others 2009b). In A. oryzae, the ability to
form CPA is strain-dependent (Chang and others 2009a); while
the cluster is complete in the NBRC 4177 strain, the RIB40
strain is unable to form the mycotoxin due to a truncation of the
PKS–NRPS (Shinohara and others 2011). Studies performed by
Liu and Walsh (2009a, 2009b) demonstrated that cpaS encodes a
PKS–NRPS responsible for cAATrp formation, while cpaD en-
codes a cAATrp-dimethylallyltransferase, leading to β-CPA. In this
cluster, the cpaO gene encoding a monoamine oxidase would lead
to CPA, thus correlating well with the previously proposed CPA
biosynthetic pathway. The study by Chang and others (2009b)
showed that disruption of this gene (named maoA in the study)
abolished the production of CPA in A. flavus.
Concerning mycotoxin biosynthetic pathways, the already de-
scribed gene clusters as well as the growing access to complete
fungal genomes will allow identifying the corresponding pathways
in cheese-related fungi. Moreover, the presence of mycotoxin-
related genes in the subphylum Mucoromycotina, so far considered
as a nonmycotoxin-producing group, could be evaluated.
Factors Modulating Mycotoxin Biosynthesis in Cheese
It is generally accepted that fungal growth and mycotoxin pro-
duction in food is influenced by multiple factors including abiotic

C 2014 Institute of Food Technologists®
Filamentous fungi and mycotoxins in cheese . . .
Figure 2–Main biotic and abiotic factors modulating mycotoxin production in cheese.
and biotic factors (Filtenborg and others 1996). In cheese, biotic
factors modulating mycotoxin production include the intrinsic ca-
pacity of a given mold species or strain to produce mycotoxins,
its physiological state and the interactions that may occur between
this organism and the other members of the cheese microbiota
(Figure 2). The main abiotic factors that are likely to modulate
mycotoxin production in cheese can be divided into manufactur-
ing and environmental factors such as temperature, relative hu-
midity (RH), ripening time, and storage atmosphere used during
cheesemaking, as well as physicochemical factors of the cheese it-
self, including chemical composition (carbon and nitrogen sources,
C/N ratio, NaCl content . . . ), pH, water activity (aw ), and redox
potential (E°) (Figure 2). Because over 1000 cheese varieties exist
worldwide, it is difficult to draw a general picture of abiotic fac-
tor variations in cheeses. For example, in surface-ripened cheeses
comprising soft, semihard, and hard types, cheeses are ripened at
an RH ranging from 90% to 80% to 85% and at temperatures of
4 to 20 °C for 14 to 540 d depending on cheese type (Robinson
1995). Moreover, pH, NaCl, and aw at the cheese surface layer
may vary at the end of ripening from 7% to 5.6%, 2% to 0.6%,
and 0.98% to 0.95%, respectively (Robinson 1995).
The physical and chemical characteristics of cheeses are also
subjected to both spatial and temporal changes during ripening.
First, environmental conditions used in cheese manufacture, such
as RH and temperature in ripening rooms and biological activities

C 2014 Institute of Food Technologists® Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 447
(glycolysis, proteolysis, and lipolysis) of cheese microorganisms,
affect physicochemical cheese characteristics during ripening.
Moreover, cheese is also characterized by a heterogeneous physic-
ochemical composition with gradients in pH, NaCl, moisture, aw ,
O2 level, and so on from the surface to the core. For example,
in the core and surface of the blue-veined cheese Danablu, pH,
NaCl, and aw gradients ranged from 4.6% to 4.4%, 1% to 6.0%
and 0.99% to 0.87%, respectively, after 1 week of ripening while
they ranged from 6.4% to 5%, 2% to 4.5% and 0.94% to 0.91%,
respectively, after 5 wk of ripening (Cantor and others 2004). Also
in Danablu cheese, van den Tempel and others (2002) indicated
that average oxygen saturation decreased from 63% to 0.1% at the
center and from 69% to 0.1% at the cheese edges from 1 to 19
wk. It is also interesting to note that in the same study, after 3 wk
of ripening, oxygen was not detected in the cheese, except in the
0.25 mm surface layer and in apparent small air pockets within the
cheese containing about 3% oxygen.
Effect of nutritional factors on mycotoxin production
While it is generally admitted that the chemical composition
of food exerts a significant effect on mycotoxin production, there
is controversy in the literature on whether cheese is a food ma-
trix that is well suited for mycotoxin production. In earlier and
much more recent studies, cheese was considered a poor substrate
for mycotoxin formation because of its low C/N ratio (Scott and
Filamentous fungi and mycotoxins in cheese . . .
others 1977; Engel 1978; Olivigni and Bullerman 1978; Finoli
and others 2001) and low ripening temperature (Marth 1979). Fi-
noli and others (2001) investigated the ability of P. roqueforti fungal
ripening cultures to synthesize roquefortine C and PR toxin in vitro
in 10% reconstituted skim milk and yeast extract sucrose (YES)
medium, which is characterized by a high C/N ratio due to its
high concentration of sucrose (150 g/L). They also analyzed, using
a HPLC-UV method, roquefortine C and PR toxin contents in 30
blue-veined cheeses from where the strains originated. The cheeses
contained very small amounts of roquefortine C (<1.44 mg/kg)
and no PR toxin was detected. Roquefortine C and PR toxin
were generated in very low quantities (<3.08 mg/L), if any, in
milk, whereas all the studied strains produced these 2 mycotoxins
in YES medium with up to 8.44 and 60 mg/L for roquefortine
C and PR toxin, respectively. In contrast, Kokkonen and oth-
ers (2005a), using LC/MS/MS, showed that roquefortine C was
present in 11 blue mold cheese samples purchased from Finnish
supermarkets at concentrations ranging from 0.8 to 12 mg/kg that
were about 10 times higher than those reported by Finoli and oth-
ers (2001). However, it is difficult to compare the concentrations
of roquefortine C reported in the latter 2 studies because different
methods were used for quantification. More importantly, different
cheese varieties likely obtained with different strains of P. roqueforti
and ripening conditions were analyzed. More studies on a large
number of blue cheese samples and varieties are therefore needed
to clarify whether or not cheese is well suited for roquefortine
C production and the relationships existing between roquefortine
C content, manufacturing practices and the toxigenic potential of
P. roqueforti strains. This observation is also true for other myco-
toxins such as CPA and MPA.
Kokkonen and others (2005b) also studied the ability of P. crus-
tosum, P. nordicum, and P. verrucosum to produce mycotoxins in 3
different substrates: YES agar as well as cheese and bread analogs.
P. nordicum, a frequent contaminant encountered in cheese, only
synthesized moderate or undetectable amount of OTA and none
of the substrates favored its production. In contrast, the substrates
had a significant impact on the secondary metabolism of the other
tested fungi. P. crustosum produced roquefortine C on all the sub-
strates, with the highest amount on the cheese analog, while it
only synthesized penitrem A on the cheese analog that was a
favorable medium for the production of this mycotoxin. The au-
thors hypothesized that the ability of P. crustosum to produce this
secondary metabolite was linked with the high protein content
of cheese because amino acids are required for the synthesis of
tremorgenic mycotoxins. In contrast, only 1 out of 8 P. verrucosum
strains produced OTA on the cheese analog, while high levels of
ochatoxin A and citrinin were produced on the bread analog. In-
terestingly, fresh goat cheese was also a more favorable substrate
for citrinin biosynthesis by P. citrinum and P. expansum than YES
medium with up to 600 mg citrinin per kg of cheese produced
after 10 d of incubation at 20 °C (Bailly and others 2002). How-
ever, as discussed by Kokkonen and others (2005b), the presence
of precursors in the substrate cannot solely explain the ability of
a given species/strain to produce higher levels of mycotoxins, and
the factors and their interrelations that modulate mycotoxin pro-
duction in food are mostly unclear. For example, in the study of
Finoli and others (2001), roquefortine C production in cheese
or milk by P. roqueforti was poor, despite the fact that the cheese
contained high protein levels including the mycotoxin precursors
(tryptophan and histidine).
In conclusion, it cannot be assumed that cheese is a poor sub-
strate in terms of its nutritional composition for mycotoxin pro-
448 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014
duction. Most probably, the ability of a mold to produce mycotox-
ins in cheese is rather species-or strain-specific. Moreover, it also
appears that the nature and the quantity of mycotoxins produced
by a given mold strain are influenced by the substrate composition
as well as some other intrinsic or environmental factors discussed
below.
Effect of NaCl on mycotoxin production
Sodium chloride in cheese contributes to flavor and acts as a
preservative through water activity reduction and microorganism
inhibition due to solubilized ions. It also enables the growth of
salt-tolerant microorganisms that mostly contribute to the sensory
properties of cheese. However, NaCl may also affect mycotoxin
production. Indeed, Schmidt-Heydt and others (2012) showed
that high amounts of OTA were produced by P. nordicum over a
wide concentration range of NaCl (5 to 100 g/L), with a weak op-
timum at 20 g/L in YES medium. A mutant strain of P. nordicum,
unable to produce OTA, displayed important growth reduction
and elevated chloride content in mycelium under elevated NaCl
concentration. It was also shown that P. verrucosum shifted from
production of citrinin to ochratoxin when NaCl concentration
was equal to or above 20 g/L with a peak at 40 g/L NaCl. As
presented above for the possible roles of mycotoxin production
in molds, these observations suggest that the biosynthesis and
excretion of OTA, containing a chloride atom, plays a role in
maintaining chloride homeostasis in the fungal cell.
Finoli and others (2001) observed that there was a positive re-
lation (r2 = 0.85) between the amount of roquefortine C and
NaCl concentration in various blue cheeses. However, due to the
number of varying intrinsic factors, it could not be extrapolated
from the latter study whether NaCl alone had an effect on myco-
toxin production by P. roqueforti. For example, Finoli and others
(2001) also found a positive relation (r2 = 0.58) between ripening
time and roquefortine C concentration. Further work is there-
fore needed to understand how the range of NaCl concentrations
commonly encountered in cheeses affect production of mycotox-
ins other than citrinin and OTA.
Effects of temperature, aw , pH, and atmosphere composition
on mycotoxin production
Whatever the fungal species (contaminant or fungal ripening
cultures), observations have been made, both in vitro and in cheese,
that mycotoxin-producing fungi are inversely correlated with tem-
perature (low or no production at refrigerated temperatures, in-
termediate production at 12 °C, and optimal production at 20
to 25 °C) (Le Bars 1979; Wagener and others 1980; Chang and
others 1991; Sweeney and Dobson 1998; Finoli and others 2001;
Taniwaki and others 2001; Bailly and others 2002; Erdogan and
others 2003). This is not surprising, since low temperature also af-
fects fungal growth that is often, but not systematically, correlated
with mycotoxin production.
Very little information is available on the effects of aw and pH on
mycotoxin production by cheese-related fungi (ripening cultures
and common contaminants, see “Filamentous Fungi in Cheeses:
The Good and the Bad” section). Most fungi able to grow in
cheese are salt-tolerant and can grow at relatively low aw con-
ditions. In P. verrucosum, OTA production in YES medium was
enhanced at both near-optimal and under mild pH and aw stress
conditions (Schmidt and others 2008). However, this does not
mean that mycotoxin production did not occur with other con-
ditions since production also occurred at other pH and aw values
(Schmidt and others 2008). In P. roqueforti, the optimum pH for

C 2014 Institute of Food Technologists®
Filamentous fungi and mycotoxins in cheese . . .
eremofortin C and PR toxin production was around 4.0 in mod-
ified YES medium (Chang and others 1991). However, no infor-
mation is currently available on the effect of aw on the production
of these secondary metabolites or other secondary metabolites
such as roquefortine C. More studies are also needed regarding
the influence of these factors on mycotoxin production by the
most common ripening cultures (P. camemberti, P. roqueforti, F.
domesticum, S. flava, and S. fusca) and contaminants (P. commune,
P. palitans, P. nalgiovense, and P. nordicum) mentioned in “Filamen-
tous Fungi in Cheeses: The Good and the Bad” section.
Gas composition (CO2 and O2 levels) of the atmosphere may
also exert a significant impact on mycotoxin production by cheese-
related fungi (Taniwaki and others 2001, 2009, 2010). Modified
atmosphere packaging (MAP), which combines the inhibitory
effect of low oxygen and elevated carbon dioxide levels, is in-
creasingly used as a hurdle technology to prevent spoilage fungi to
grow in cheese (Haasum and Nielsen 1998). Taniwaki and others
(2001) compared mycotoxin production by strains of A. flavus,
P. roqueforti, and P. commune on sliced-Cheddar cheese in air and
the combined presence of CO2 (20% or 40%) and O2 (1% or 5%).
Mycotoxin production was greatly decreased under modified at-
mosphere conditions compared to production in air. For example,
roquefortine C and CPA production were divided by factors of
7- to 20-fold and 12- to 850-fold under modified atmospheres
as compared to mycotoxin production in air by P. roqueforti and
P. commune, respectively. On the other hand, the gas composi-
tion in the cheese core may also influence secondary metabolite
production by P. roqueforti during blue-cheese ripening. In blue-
veined cheeses such as Danablu cheese, O2 levels decrease rapidly
during the 1st weeks of ripening, while the highest CO2 level re-
ported in Danablu cheese was 20% (van den Tempel and Nielsen
2000). Consequently, these atmosphere conditions are most likely
unfavorable for mycotoxin production in cheese.
Effects of biotic factors
To our knowledge, the intraspecific mycotoxigenic potential of
fungal species found in cheese has not yet been extensively studied.
Nevertheless, the toxigenic potential of different strains of P. roque-
forti and P. camemberti was highly variable (Le Bars 1979; Chang
and others 1991; Finoli and others 2001). As already mentioned,
the quantity and nature of secondary metabolites produced may
be a strain-dependent trait. For example, high intraspecific differ-
ences in MPA/PR toxin ratios have been reported in P. roqueforti
(O’Brien and others 2006).
Microbial interactions play an important role in cheese quality
and safety (Irlinger and Mounier 2009). Cheese microbiota or the
addition of protective cultures may provide a hurdle effect (com-
petitive interactions, production of inhibitory compounds) toward
potentially toxigenic fungal contaminants that, in turn, are unable
to produce mycotoxins (Nielsen and others 1998; Irlinger and
Mounier 2009; Dalié and others 2010). Finally, it is worth noting
that very little attention has been given to studying the effects of
cheese cultures with beneficial use on mycotoxin production by
fungal ripening cultures.
As mentioned above, further work is still required to fully under-
stand the effect of biotic and abiotic factors on fungal growth and
subsequent mycotoxin production in cheese. The effects of single
abiotic factors and their interactions on mycotoxin formation and
the intraspecific variability within mycotoxigenic spoilage fungi
and fungal ripening cultures deserve further attention. With the
development of mathematical models able to predict fungal growth
and mycotoxin production, the cheese industry will be able to ap-

C 2014 Institute of Food Technologists® Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 449
ply them to both existing and newly developed products. This will
allow companies to better define manufacturing processes, storage
conditions, shelf-life, and will contribute to the foodborne my-
cotoxin risk assessment (Membré and Lambert 2008; Garcia and
others 2009).
Control of Mycotoxins in Cheeses
As previously stated, some authors consider that cheese is very
susceptible to mold growth as well as mycotoxin production
(Sengun and others 2008). On the contrary, other authors
(Gourama 1997) have indicated that conflicting reports can be
found in the literature concerning mycotoxin production in cheese
and dairy products, and concluded that cheese is actually a better
medium for mold growth than for mycotoxin production. The
impact of toxic molds on cheese is relatively low and nontoxic
molds overgrow toxic ones; moreover, cheese is a poor substrate
for mycotoxin production if correctly stored at a low temperature
(5 to 7 °C) (Bullerman 1981). However, even if the risk is con-
sidered to be low, it should be controlled. The 1st goal is to avoid
mycotoxins in milk. Then, during cheese production and storage
until consumption, 2 different aspects should be considered, fungal
contaminants that potentially produce mycotoxins are undesirable
and should be avoided and fungal ripening cultures intentionally
added during cheesemaking (for example, blue-veined cheeses or
camembert-type cheeses) should not be mycotoxigenic.
Control of mycotoxins in milk
Good animal feeding practices are necessary to avoid mycotoxins
in feed products and subsequently, in milk (mainly AFM1). Molds
able to produce mycotoxins may grow on plant materials in the
fields before harvest, during handling and storage, as well as during
transformation into feed products (Jouany 2007).
During harvest and postharvest steps, mycotoxin control in-
cludes: early harvest usually associated with low concentrations of
mycotoxins, high cutting heights to avoid contamination from soil
particles and elimination of damaged grains that can favor fungal
growth, low humidity during storage (the main factor to avoid
fungal growth, critical limit of 14% w/w), and cool storage tem-
peratures and ventilation that are important secondary factors to
reduce the growth risk (Jouany 2007).
For silage, humidity remains relatively high; therefore, is it nec-
essary to respect the optimal moisture content at harvest (33%
to 35% dry matter for the entire plant [Demarquilly 1994] and
use good agricultural practices when filling silos). By following
these guidelines, anaerobic conditions are encountered and lactic
fermentation takes place thus avoiding fungal growth.
LAB, known for their antifungal activities, can be inoculated
during silage production and are now more frequently used than
chemical preservatives (Kalac 2011). A Lactobacillus casei subsp. pseu-
doplantarum 371 isolate from silage, was studied for its ability to
inhibit mold growth and A. flavus subsp. parasiticus NRRL 2999
AF production (Gourama and Bullerman 1997). Numerous bio-
logical additives, usually containing LAB, are currently marketed
for silage control. However, the main recommendations for usual
practice are to quickly create stable anaerobic conditions and to
apply propionic acid, well known for its antimold activity (Kalac
2011). In Europe, propionic acid (E code = E280) is authorized
for use in feed without a time limit and, consequently, different
acidic additives for silage containing propionic acid are currently
sold.
Concerning milk containing mycotoxins, curative physical,
chemical, and/or biological treatments to eliminate mycotoxins
Filamentous fungi and mycotoxins in cheese . . .
are poorly efficient and too expensive to bring viable solutions for
feed decontamination (Jouany 2007; Kolossova and others 2009).
Different physical and chemical methods have been recommended
to detoxify food and feed; however, only a few (ammonia treat-
ment to destroy AF for instance) are practically used (Bata and
Lásztity 1999). Up to 95% to 98% of AF is decomposed by am-
moniation that is consequently used in various countries for animal
feedstuffs decontamination (van Egmond 2004).
Milk-producing animals are more or less efficient barriers
against mycotoxins. Rumen microorganisms and the liver pro-
duce toxic metabolites (Kalac 2011). However, of healthy animal
rumen microbial communities also contribute to prevent health
risks as they biotransform different mycotoxins (for instance, de-
oxynivalenol or zearalenone, the main mycotoxins produced in
silage) into harmless metabolites. Consequently, milk usually con-
tains low concentrations of mycotoxins.
Mycotoxins are generally stable compounds. However, heat
treatment and ultraviolet light application have been shown to
be efficient methods to reduce AFM1 content in milk (Rustom
1997).
Control of mycotoxigenic fungal contaminants in cheese
Good hygiene and sanitation standards throughout the entire
food chain permit to minimize mold presence and growth on
cheese (Bullerman 1981).
Cold storage. Cold storage (5 to 7 °C) is an important step
in mycotoxin prevention (Sengun and others 2008). Penicillium
species are the only molds growing at such temperatures (Buller-
man 1981) and are less problematic as they do not produce AFs or
STC. Such species could produce less toxic and less stable metabo-
lites in cheese (such as MPA). At the consumer level, cheeses
should not be maintained at room temperatures unless other pre-
vention techniques are employed.
Chemical preservatives. Chemical preservatives can be used to
control mycotoxins (Sengun and others 2008). For instance, pi-
maricin (= natamycin, E code = E235, produced by the acti-
nobacterium Streptomyces natalensis) was shown to delay fungal
growth and as a consequence to avoid mycotoxins (Kiermeier and
Zierer 1975; Nilson and others 1975). Its direct effect on produc-
tion of mycotoxins in cheese remains poorly documented. This
antifungal agent is now permitted and used in cheese in Europe
and the United States. Private companies sell natamycin formu-
lations for use in cheese production. In Europe, the admissible
daily intake value is 0.3 mg/kg of body weight. Mold growth was
inhibited in vacuum-packed Kashar cheese containing natamycin,
for a 5-mo ripening period at 4 °C (Var and others 2006).
Preservatives such as sorbic, benzoic, and propionic acids can
inhibit mold growth, except for P. roqueforti. This species is resistant
and, consequently, the most important spoilage species, except
for blue-veined cheeses (Filtenborg and others 1996). The U.S.
Federal Standard of Identity permits the use of sorbate in cheeses
at levels under 0.3%, calculated as sorbic acid (Sengun and others
2008). Propionates are permitted for the surface treatment (such
as wrapping materials) of cheese and cheese products (Nielsen and
de Boer 2004).
Mold growth on cheeses can also be reduced using high sodium
chloride concentrations that decrease water activity required for
growth and mycotoxin production (Sengun and others 2008);
however, both organoleptic factors and health considerations have
to be taken into account in this context.
Reduction or absence of oxygen. Since mycotoxin-producing
molds are obligate aerobes, mold growth and mycotoxin forma-
450 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014
tion are minimized by low oxygen concentrations and/or high
concentrations of other gases (CO2 ) in the gaseous phase (Buller-
man 1981). Consequently, vacuum-packaging is used to inhibit
fungal growth, and in such conditions, production of roquefortine
C and CPA by P. roqueforti and P. commune is virtually eliminated
(Taniwaki and others 2001).
Plant extracts. Knowing their organoleptic characteristics, an-
timicrobial herbs, spices, or their essential oils have long been
used to prevent fungal growth in cheese (Sengun and others
2008). On this basis, eugenol and thymol were shown to affect
growth and citrinin production by 2 P. citrinum strains in Spanish
cheeses (Vázquez and others 2001). Eugenol appeared more effi-
cient than thymol and the effects reported were cheese-dependent.
Fungal growth was totally inhibited in Arzu´a-Ulloa cheese with
200 mg/mL of eugenol, but no effect was observed in Cebreiro
cheese. At lower concentrations, permitting fungal growth, cit-
rinin production inhibition appeared to be limited or even absent.
Microbial control. Competing and/or inhibiting microorgan-
isms can also limit fungal growth and mycotoxin production
(Sengun and others 2008). When fully viable, the secondary
ripening cultures P. camemberti and G. candidum can efficiently
compete against contaminants (Nielsen and others 1998; Decker
and Nielsen 2005). Species belonging to the Lactococcus and Lac-
tobacillus genera are the most capable of preventing or limiting
mycotoxigenic mold growth because of organic acid production,
competition for nutrients, and the production of other antago-
nistic compounds (Dalié and others 2010). Raw milk from ewe,
cow, and goat was shown to be a productive reservoir for antifun-
gal bacteria, most of them belonging to Lactobacillus (Delavenne
and others 2011). Eleven antifungal Lactobacillus species showed an
antifungal activity in milk with strain-dependent activity spectra
(fungal species tested were Debaryomyces hansenii, Kluyveromyces lac-
tis, Kluyveromyces marxianus, Penicillium brevicompactum, Rhodotorula
mucilaginosa, and Yarrowia lipolytica). Lb. harbinensis showed very
strong antifungal effect in yogurt inhibiting all tested fungi. Lacto-
bacillus amylovorus DSM 19280 was shown to inhibit Penicillium
expansum, P. roqueforti, Aspergillus niger, Aspergillus fumigatus, and
Fusarium culmorum. In the presence of the bacterial adjunct, ched-
dar cheeses exposed to natural airborne fungi benefited from a
6-d delay in the appearance of mycelia on their surface without
detectable negative impact on cheese quality (Lynch and others
2014).
Dairy propionibacteria, widely used for Swiss-type cheese pro-
duction, are known to ferment glucose, lactic acid, and glycerol
into propionic acid, acetic acid, and carbon dioxide. Antifungal
effect of dairy propionibacteria was studied using glycerol as car-
bon source for bacterial growth. Five type strains of propionibacteria
were tested against the yeast Rhodotorula mucilaginosa and the molds
Penicillium commune and Penicillium roqueforti. Increase in glycerol
concentrations enhanced the inhibition of the molds when the
yeast was less affected. This effect was shown to be due to the
production of propionic acid and simultaneous pH reduction of
the medium (Lind and others 2010).
Efficient biocontrol solutions for cheesemakers could likely
emerge from those recent scientific results.
Selection of nontoxigenic ripening cultures
Mold ripening cultures used to produce mold-ripened cheeses
should have low toxigenic capacity (Sengun and others 2008).
Variability within the P. camemberti species in regards to toxigenic
capacity allows for the selection of weakly toxicogenic strains (Le
Bars 1979). In pure cultures, all known P. camemberti strains are

C 2014 Institute of Food Technologists®
Filamentous fungi and mycotoxins in cheese . . .
able to produce CPA (Nout 2004); however, it is possible to select
CPA-negative mutants. Consequently, selected ripening cultures
were CPA-negative and only very low CPA levels were detected
in Camembert-type cheeses. The same observations concerning
mycotoxin production variability by P. roqueforti have been made
(Finoli and others 2001). It was concluded that roquefortine C
was the only metabolite found and that its low toxicity com-
bined with the low levels observed make blue cheese consump-
tion safe for consumers. Moreover, mycotoxin production can be
stopped by gene inactivation of the biosynthetic pathway using
mutation/selection processes or genetic engineering (Geisen and
Holzapfel 1996). However, one should keep in mind that such
genetically modified organism (GMO) ripening cultures are not
socially accepted everywhere (Costa-Font 2011) and that special
regulations could apply to the obtained products.
Common sense advice has also been expressed (Sengun and oth-
ers 2008): to avoid the mycotoxin risk, cheeses with spontaneously
growing molds should not be consumed.
To conclude on mycotoxin control in cheeses, we can recall
that good practices in animal feeding are available to avoid myco-
toxin presence in milk, and good hygiene and sanitation standards
during milking, milk storage, and cheese production are required.
These guidelines associated with efficient selection and prepara-
tion of ripening cultures, cold storage of cheese, as well as adapted
packaging can all prevent growth of fungal contaminants.
Conclusion
Filamentous fungi encountered in cheeses may originate from
raw materials such as milk or may be introduced during cheese-
making either from the environment (contamination), or de-
liberately inoculated using commercial ripening cultures. Con-
tamination by filamentous fungi can be detrimental to cheese
quality, causing appearance defects, off-flavors, and/or potentially
toxic secondary metabolite production including mycotoxins. My-
cotoxin production profiles may change under different fungal
growth conditions, including substrate composition, ecophysio-
logical factors (temperature, water activity, pH, or oxygen con-
centration), and biotic factors (strain-dependent mycotoxin pro-
duction). To detect a wide spectrum of mycotoxins in cheese,
numerous techniques are currently available. Due to the high com-
plexity of the cheese matrix, LC/MS/MS seems to be the most
sensitive technique currently available to quantify mycotoxins in
cheeses.
Concerning mycotoxin toxicity, for those present in cheese,
there is a lack of recent scientific data. The most common myco-
toxins stable in cheese are AF, citrinin, CPA, roquefortine C, STC,
and MPA. Others, including patulin, penicillic acid, and PR toxin
do not persist in cheese due to the microaerophilic conditions
encountered. Toxicological investigations must be made with new
tools to understand the toxicity mechanisms of these mycotoxins.
Further knowledge on mycotoxin biosynthetic pathways should
help to develop methodologies to elucidate the ecophysiological
roles and control mycotoxin production in cheeses. The molecular
basis of these pathways may also lead to complementary approaches
for mycotoxin control. These guidelines, associated with efficient
ripening cultures, selection of nontoxigenic strains, cold storage as
well as adapted packaging conditions can all prevent the growth of
fungal contaminants. This review emphasizes on future challenges
that need to be addressed by the scientific community, fungal cul-
ture manufacturers, and artisanal and industrial cheese producers.

C 2014 Institute of Food Technologists® Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 451
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